Bio 397 - Bioinformatics Final Exam

what is the principle of operation of Sanger technology

chain termination

What is Chain Termination?

Uses fluorescently labeled ddNTPs to stop DNA synthesis @ specific bases

• ddNTPS = dideoxyribonucleoside triphosphates = modified DNA building blocks

what is the principle of operation of Illumina technology

sequencing by synthesis

What is Sequencing by Synthesis?

Reversible terminator fluorescent nucleotides are added; a camera records the "flash" of each base.

what is the principle of operation of PacBio technology

SMRT (Single Molecule Real Time)

What is SMRT (Single Molecule Real Time)?

Uses a ZMW (Zero-Mode Waveguide) to observe a single polymerase incorporate bases in real-time

what is the principle of operation of MinION technology

nanopore

What is Nanopore?

Measures changes in electrical current as a single-stranded DNA molecule passes through a protein pore

what uses fluerescently labled ddNTPs to stop DNA synthesis at specific base

Sanger

what uses reversible terminator fluorescent nucleotides are added; a camera records the "flash" of each base

Illumina

what uses a ZMW (zero ode waveguide) to observe a single polymerase incorporate bases in real-tine

PacBio

what measures changes in electrical current as a single-stranded DNA molecule passes through a protein pore

MinION

what are pros to sanger technology

very high accuracy, long reads (800bp)

what are cons to sanger technology

low throughput, very expensive per base. used for single genes/plasmids

what are pros of illumina technology

massive throughput (billions of reads), lowest cost per base, very accurate

what are cons of illumina

very short reads (150-350bp), difficult to assemble repetitive regions

what are pros of pacbio

long reads, can span repetitive regions and detect base modifications

what are cons of pacbio

historically higher error rates, lower throughput than illumina, higher cost

what are pros of minion

ultra long reads, real time data, highly portable (USB)

what are cons of minion

high error rate compared to illumina; requires high-quality dna input

De Novo Assembly

the process of taking short, overlapping DNA reads and stitching them together into contigs (contiguous sequences) without a reference genome

what type of reads make assembly easier

longer reads (PacBio/MinION)

why do longer reads make assembly easier?

they bridge repetitive "dead zones" that short illumina reads cannot

True or False: 200bp for Illumina sequencing is good as it only produce the
short reads but for PacBio it is low quality as the sequencing technique always produce
longer reads.

True

Ambiguous Bases

Represented by the letter N. A high "N" count indicates areas
where the sequencer couldn't determine the base. Lower is better.

what does a high N count indicate

areas where sequencer couldnt determine the base

what type of N count is better

lower

what is the PHRED Quality Score (Q)

measure of the quality of the identification of the nucleobases

what is the standard benchmark for "high quality"

Q30 (>99.9% accuracy)

Coverage (Depth)

the average number of times a base is sequenced

What does a higher coverage increase?

confidence in consensus sequence

Contig Length & N50

• statistical measure of assembly "contiguity."

• It is the length of the shortest contig such that all contigs of that length or longer sum to 50% of the total assembly

N50 is a statistical measure of…

assembly "contiguity"

What type of N50 indicates a better assembly?

higher N50

What occurs due to repetitive sequences or regions that are difficult to sequence?

gaps

• ex: high GC content

What are the 2 things used to close gaps?

Primer Walking & Hybrid Assembly

Primer Walking

designing specific primers at the end of a contig & using Sanger sequencing to "walk" into the gap

Hybrid Assembly

Combining Illumina (for accuracy) with PacBio/MinION (to span long gaps)

What gap-closing method is normally used & why?

Hybrid Assembly

• cheap & efficient

What are the 2 main strategies for Hybrid Assembly?

Short-read First (Scaffolding) . . . OR . . .Long-read First (Polishing)

Short-read First (Scaffolding)

1. You assemble the highly accurate Illumina reads into contigs.

2. You then use the Long Reads as "bridges" to tell the assembler which contigs sit
   next to each other, creating a scaffold.

Step 1 in short-read first (scaffolding)

assemble highly accurate illumina reads into contigs

Step 2 in short-read first (scaffolding)

use long reads as "bridges: to tell assembler which contigs sit next to each other, creating scaffold

Long-read First (Polishing)

1. You assemble the genome using only the Long Reads. This creates a very
   "complete" genome (few gaps) but with many small "typos" (Indels).

2. You then map the Short Reads onto that assembly to "polish" it—correcting
   the 1% error rate of the long reads with the 99.9% accuracy of Illumina.

Step 1 in long-read first (polishing)

assemble genome using only long reads

Step 2 of long-read first (polishing)

map short reads onto that assembly to "polish" it

.

.

What is the role of a polisher in Hybrid Assembly?

fixes typos

What is the role of the bridge in Hybrid Assembly?

spans the repeats

Short Reads (Illumina) Overview

• Accuracy: High (Q30+)

• Contiguity: Fragmented (many gaps)

• Cost: Cheap

• Role: Polisher

Long Reads (PacBio/Nanopore) Overview:

• Accuracy: lower

• Contiguity: High (can produce “closed” genomes)

• Cost: Expensive

• Role: Bridge

Node

represents a common ancestor where lineage splits

Branches

the evolution of lineage over time

Tips (leaves)

represent the existing taxa (species/sequences) being compared

Clade (monophyletic group)

a group consisting of a common ancestor and all descendants

Root

the common ancestor of all sequences in the tree

Branch Length

the horizontal length correlates with the amount of genetic change (mutations) over time

What does the horizontal length correlate with in a phylogenetic tree?

the amount of genetic change (mutations) over time

Sister Taxa

2 lineages that emerged from same immediate node

Transcriptomics

the study of the transcriptome— the sum of all RNA transcripts in a cell

What is the sum of all RNA transcripts in a cell?

transcriptome

Transcriptomics: Microarray

• Detection: hybridization — pre-designed probes

• Novelty: can only detect known genes on the chip

• Dynamic Range: low (signals saturate/are too weak)

• Cost: fixed per sample

Trancriptomics: RNA-Seq

• Detection: sequences all cDNA

• Novelty: can discover new transcripts, isoforms, & non-coding RNAs

• Dynamic Range: high (can detect low & very high expression)

• Cost: depends on sequencing depth

Microarrary uses what type of probes?

pre-designed (hybridization)

Microarray can only detect what type of genes?

known genes on the chip

What is the cost of Microarray?

fixed per sample

RNA-seq has what type of sequencing?

direct sequencing (sequences all cDNA)

What type of method can discover new transcripts, isoforms, and non-coding RNAs?

RNA-seq

The logic that more mRNA molecules in the sample means…?

more cDNA produced

The logic that more cDNA is produced means…?

more reads mapped to that gene during sequencing

what is often used for differential expression?

log fold change (LogFC)

What does Log2FC of 1 mean? Of -1? Of 0?

• 1: expression has doubled

• -1: expression has halved

• 0: expression is the same

What is the concept of bulk RNA-seq?

take a whole piece of tissue —> grind it up —> sequence total RNA

What do you get from bulk RNA-seq?

an average expression profile for entire sample

What are the pros of bulk RNA-seq?

• High sensitivity for low-abundance transcripts,

• Cost-effective

• Robust for comparing “Condition A” vs “Condition B”

What is the con of bulk RNA-seq?

Masks heterogeneity.

• if a rare cell type is the only one expressing a gene, its signal might be drowned out by the “average”

What is the concept of Single-Cell RNA-seq / scRNA-seq?

dissociate the tissue into individual cells & “barcode” RNA from each cell before sequencing

What do you get from scRNA-seq?

list of every gene expressed in every individual cell

What are the pros of scRNA-seq?

• Can identify rare cell types

• Allow for Pseudotime analysis (tracing how a cell changes over time, like during development or cancer progression…)

What are the cons of scRNA-seq?

• You lose the "where":

  • Once you dissociate the cells, you don't know which cell
    was sitting next to which

• Prone to “dropout”

  • a gene is expressed but the sequencer misses it b/c starting material is so small

What is the concept of Spatial Transcriptomics?

You sequence the RNA while it is still attached to a thin slice of the tissue, using a slide with "spatial barcodes" that record coordinates

What do you get from Spatial Transcriptomics?

gene expression data mapped directly onto physical structure of tissue

Pros of Spatial Transcriptomics

• Crucial for understanding microenvironments

  • (e.g., how immune cells interact
    with the edge of a tumor).

• Provides "geographic" context that scRNA-seq lacks

Cons of Spatial Transcriptomics

• Expensive

• Many platforms have lower resolution

  • (each "spot" might contain 5–10 cells rather than just one)

Bulk RNA-Seq overview

• Resolution: Tissue level (average)

• Cell Heterogeneity: Hidden/masked

• Spatial Context: Lost

• Best For: General biomarkers, comparing treatments

Single-Cell RNA-seq (scRNA-seq) overview

• Resolution: Cellular level

• Cell Heterogeneity: Fully visible

• Spatial Context: Lost

• Best For: Finding new cell types, developmental lineages

Spatial RNA-seq overview

• Resolution: Tissue architecture

• Cell Heterogeneity: Visible in context

• Spatial Context: Preserved

• Best For: Tumor microenvironments, brain mapping